DETECTION OF SOME OF VIRULENCE GENES IN SALMONELLA KENTUCKY ISOLATED FROM POULTRY

Assiut University web-site: www.aun.edu.eg

DETECTION OF SOME OF VIRULENCE GENES IN SALMONELLA KENTUCKY ISOLATED FROM POULTRY

MONA A. A. ABDEL RAHMAN¹; SOAD A. NASEF²  and ENGY A. HAMED³

¹ Reference Laboratory for Veterinary Quality Control on Poultry Production, Nadi El-Seid

Street, Dokki P.O. Box 246, Giza 12618, Egypt, Email: Drmonaali2000@yahoo.com

² Animal Health Research Institute, Nadi El-Seid Street, Dokki P.O. Box 246, Giza 12618, Egypt

³ Ministry of Agriculture, Nadi El-Seid Street, Dokki P.O. Box246, Giza 12618, Egypt

Received: 28 June 2017;       Accepted: 30 July 2017

INTRODUCTION

Salmonella is a major zoonotic pathogen in Europe, causing approximately 152,000 confirmed human infections in 2007 (Anonymous, 2009).

Salmonellosis is world wide spread among human and animals caused by different serovars belong to Salmonella enterica subspp. Enterica as Enteritidis, Typhimurium, Newport, and Javiana, Salmonella Kentucky ST198 has an increasing multiple drug resistant which consequently showing it has a public health hazard (LeHello et al., 2011).

Food and Safety Inspection Service (FSIS), from 2000 to 2009 reported that Salmonella Kentucky, Enteritidis, Heidelberg and Typhimurium are commonly found in broilers and ground chicken (Andino and Hanning, 2015).

Most virulence genes of Salmonella are clustered in regions distributed over the chromosome called  Salmonella pathogenicity islands (SPI)  (van Asten and van Dijk, 2005).

Corresponding author: Dr. MONA A. A. ABDEL RAHMAN

E-mail address:Drmonaali2000@yahoo.com

Present address: Reference Laboratory for Veterinary Quality Control on Poultry Production, Nadi El-Seid Street, Dokki P.O. Box246, Giza 12618, Egypt

These gene-clusters might be acquired by Salmonella from other species through horizontal gene transfer, this hypothesis is based on the significant difference in GC content of the islands compared to that of the residual genome and the remnants of bacteriophages or transposon insertion sequences that often mark the borders of the islands (van Asten and van Dijk, 2005).

Amplification of invA gene now has been recognized as an international standard for detection of Salmonella genus (Malorny et al., 2003).Invasion gene (invA) responsible for intestinal mucosa invasion by all Salmonellae (Fluit, 2005; Chuanchuen et al., 2010), this gene which is chromosomally located aids attachment of the pathogen to the epithelial cells (Galán and Curtiss, 1989).

The Sop proteins (sopA-E) (sop) and the heat-labile Salmonella enterotoxin (stn) are effector proteins that is integrated in pathogenesis of Salmonella through survival and replication (van Asten and van Dijk, 2005).

There are several virulence factors contributing to Salmonella adhesion and invasion mechanism, as Salmonella plasmid virulence (spv) operon, which consists of five genes (spvRABCD), potentiates the systemic spread of the pathogen and aids in its replication in extra-intestinal sites (Zou et al., 2012). spvR is a positive regulatory protein essential for the expression of the other spv genes (Guiney et al., 1995) while spvB product ADP-ribosylates actin, make imbalance of  the eukaryotic cell (Lesnick et al., 2001). Experiments have shown that spvB together with spvC is responsible  for virulence to Salmonella Typhimurium when administered subcutaneously to mice (Matsui et al., 2001).

mgtC gene encodes MgtB Mg2+ transporter and helps Salmonella  to survive within macrophages (Alix and Blanc-Potard, 2007).

The sigDE operon encodes SigD (SopB), a multifaceted effector that is involved in many steps of pathogenesis  (Fàbrega and  Jordi, 2013).

While the fimbrial gene bcfC has a role in cell invasion (Huehn et al., 2010). Induction of cell apoptosis to limit the host’s inflammatory responses is mediated by the avrA gene (Borges et al., 2013).

Aim of this work is to determine the extent to which virulence genes (invA, avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, spvC, sopE1, bcfC) existence in Salmonella Kentucky from avian origin that may pose a risk to the human population and poultry in Egypt.

MATERIALS AND METHODS

Bacterial isolates:

Collection of Salmonella Kentucky strains from July 2011 to May 2016 was done during routine examination of different samples submitted to reference laboratory for veterinary quality control on poultry production, a total of 26 Salmonella Kentucky strains were collected from internal organs, feed, embryonated eggs, drag swabs and paper lining chick box, twenty two from chicken and four from quail.

Bacterial isolation and identification:

The detection and identification of Salmonella isolates was done according to ISO 6579/cor.1.2004 and by serotyping of all the Salmonella isolates were done by slide agglutination using commercial O and H antisera (Difco Laboratories, Detroit, MI, USA) in accordance with the Kauffmann–White typing scheme and ISO/TR 6579-3:2014.

Polymerase chain reaction (PCR):

DNA extraction was performed using QIAamp DNA mini kit (Qiagen, Germany, GmbH Catalogue no.51304).

Oligonucleotide primer: primers were used supplied from metabion (Germany) and PCR conditions was mentioned as in Table (1).

All samples were confirmed by using Conventional PCR technique by using invA gene.

Virulence gene detection:

Conventional PCR technique was used for detection of virulence determinants by detection of 10 virulence genes (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) in the 26 Salmonella Kentucky strains by conventional PCR technique.

Isolates were purified on LB (luria-Bertani) agar and subsequently grown overnight at 37°C in LB broth.

PCR amplification: a volume of 25 µL PCR reaction containing 12.5 µL of Emerald Amp Max PCR Master Mix (Emerald, Japan), 1 µL of each primer of 20 pmol concentrations, 4.5 µL of Depic water and 6 µL of template was used in a Biometra thermal cycler. The reference strains provided by the External Quality Assurance Services (EQAS) were used as positive controls of S. Kentucky. DNA of the negative control (E. coli NCIMB 50034).

Analysis of the PCR products:

The PCR products were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V\cm. for gel analysis 5 µL of the products was loaded in each gel slot. Gene ruler 100-1000 bp ladder. Thermo scientific was used to determine the fragment size. The gel was photographed using a gel documentation system (applied).

Table 1: Virulence factor targets and primers, including nucleotide sequences, PCR conditions, and references.

^a   PCR was done for 35 cycles.

^b   After 30 cycles, final extension step of 4 minutes at 72ºC was performed.

^c SCV, Salmonella-containing vacuole.

RESULTS

1. Bacterial isolates:

A total of 26 Salmonella Kentucky strains; twenty two from chicken (2 drag swabs, 1 table eggs, 1 embryonated eggs, 6 internal organs, 2 feed, 10 paper lining chick box) and four from cloacal swabs from  quail.

2.  Virulotyping:

All isolates were screened using PCR analysis for the presence or absence of 11 selected virulence genes (Table 1). The invA were detected in 100% of the Salmonella strains; SopB 92.3%. 88.4%  of  strains were carry, avrA, bcfC and ssaQ, mgtC (80.7%), sopE and sodC (19.2%), siiD (11.5%), spvC (3.8%),  while no one carry gipA.

Table 2: Distribution of the virulence genes among Salmonella Kentucky strains.

Photo (1). Agarose gel electrophoresis showing Salmonella specific PCR of Salmonella Kentucky using primer set for the invA (284 bp) gene

Lanes 1 - 19:  positive samples of Salmonella Kentucky

Lane 20: Negative control (E. coli NCIMB 50034)

Lane 21: Positive control (Salmonella KentuckyEQAS)

Lane 22: DNA ladder.

Photo (2): Agarose gel electrophoresis showing Duplex PCR with amplification of 422 bp and 677 bp bp fragments for avrA and mgtC genes of Salmonella Kentucky performed with their specific primers

Lane 1: DNA ladder

Lane 2: Negative control (E. coli NCIMB 50034)

Lane 3: Positive control (Salmonella KentuckyEQAS)

Lanes 4-10: positive Salmonella Kentucky samples for avrA and mgtC genes         

Photo (3): Agarose gel electrophoresis showing Duplex PCR with amplification of 455bp and 655bp fragments for ssaQ and siiD genes of Salmonella Kentucky performed with their specific primer

Lanes 1: DNA ladder

Lane 2: Positive control (Salmonella KentuckyEQAS)

Lane 3: Negative control (E. coli NCIMB 50034)

Lanes 4,9: positive Salmonella Kentucky samples for ssaQ and siiD genes

Lanes 6,7,8: positive Salmonella Kentucky samples for ssaQ gene

Lane 5: Negative Salmonella Kentucky sample for ssaQ and siiD genes

Photo (4): Agarose gel electrophoresis showing PCRamplification of the 422 bp SopE1 gene of Salmonella Kentucky.

Lanes 1-5, 7, 9-14: Negative Salmonella Kentucky samples for SopE1gene

Lanes 6, 8: positive Salmonella Kentucky samples for SopE1gene

Lane 15: Positive control (Salmonella KentuckyEQAS)

Lane 16: Negative control (E. coli NCIMB 50034)

lane17: DNA ladder

Photo (5): Agarose gel electrophoresis showing PCR with amplification of 424 bp gene SodC1 of Salmonella.

Lane 1: DNA ladder

Lane 2: Negative control (E. coli NCIMB 50034)

Lane 3: Positive control (Salmonella KentuckyEQAS)

Lanes 4, 7, 8: positive Salmonella Kentucky samples for SodC1gene

Lane 5, 6: Negative Salmonella Kentucky sample for SodC1gene

Photo (6): Agarose gel electrophoresis showing PCR with amplification of 467bp fragments for spvC gene of Salmonella Kentucky performed with the specific primer

Lanes 1-10: Negative Salmonella Kentucky samples for spvCgene

Lane 11: positive Salmonella Kentucky samples for spvCgene

Lane 12: Negative control (E. coli NCIMB 50034)

Lane 13: Positive control (Salmonella KentuckyEQAS)

Lane 14: DNA ladder

Photo (7): Agarose gel electrophoresis showing amplification product of 517bp fragments of sopB gene of Salmonella Kentucky performed with the specific primer

Lanes 1-12: positive Salmonella Kentucky samples for sopBgene

Lane 13: Positive control (Salmonella KentuckyEQAS)

Lane 14: Negative control (E. coli NCIMB 50034)

Lane 15: DNA ladder

Photo (8): Agarose gel electrophoresis showing amplification product of 467bp fragments of bcfC gene of Salmonella Kentucky performed with their primer.

Lanes 1: DNA ladder

Lanes 2,4-12: positive Salmonella Kentucky samples for bcfC gene

Lane 3: Negative Salmonella Kentucky sample for bcfC

Lane 13: Positive control (Salmonella KentuckyEQAS)

Lane 14: Negative control (E. coli NCIMB 50034)

Table 3:  Distribution of virulence genes combinations in Salmonella Kentucky.

DISCUSSION

Both the presence and the dissemination of Salmonella spp. in foods represent an important issue to the poul­try industry, since they could determine a decrease in the consumption of poultry meat, posing a threat to the national and international poultry trading (Ikuno et al., 2004).

S. Kentucky is widely distributed in broiler in America more over it has isolated from poultry and poultry products and it was of high antibiotic resistance  ( Fricke et al., 2009).

S. Kentucky is isolated from several species hasn't any signs of illness as cattle, poultry, poultry products, environment and domesticated dogs in the United States (Haley et al., 2016 a) S. Kentucky ST198 is responsible for several human cases who were travel to Middle East, Southeast Asia or Africa (LeHallo et al., 2011, 2013 a,b).

Screening by PCR based on 11 well known virulence genes was applied. The results showed that variable dissemination percent among Salmonella kentucky (table 2). The results indicated that only little or no variation was found for genes incorporated in SPIs and for the fimbrial marker, which is in accordance with (Huehn et al., 2010) and assure that virulence genes are widely distributed among Salmonella serovars.

The variety of virulence factors among Salmonella serovars has resulted in differences in their pathogenicity (Fluit, 2005). The detection of invA gene in all the examined isolates is in agreement with previous reports in Egypt (Osman et al., 2013, 2014a, 2014b, Ahmed et al., 2016). and worldwide (Chuanchuen et al., 2010; Borges et al., 2013; Rowlands et al., 2014). The invA gene encodes for a protein in the inner and outer membrane, which is essential for the invasion of epithelial cells (Darwin and Miller, 1999). These studies described this gene as a marker for the molecular detection of Salmonella serotypes by PCR ( Salehi et al., 2005).

The invA gene, the sopB, bcfC, avrA,ssaQ and mgtC  genes were present in the most of strains. On contrary, the gipA gene was absent from all Salmonella strains.

Based on the PCR with 11 most important virulence genes, the virulotyping results for tested Salmonella Kentucky strains show variable results (sopB (24\26), avrA, bcfC and ssaQ (23\26), mgtC (21\26), sopE and sodC (5\26), siiD (3\26), spvC (1\26), while no one carry gipA.

In our work sopB, avrA, ssaQ, mgtC, bcfC have the highest recorded Percent of tested virulence genes that's nearly similar to (Huehn et al., 2010, Osman et al., 2014b).

avrA gene was detected also in 88.4% of the isolates. The high frequency of this gene is only observed in serovars that have a potential to cause severe salmonellosis in humans (Borges et al., 2013).

The inclusion and reassortment of such prophage-associated virulence genes may help Salmonella to change its behavior adaptation and acquire new changes. Also fimbriae are responsible for adhesion of bacterium to the cells. They are a set of fimbrial determinants (including bcf, agf, csg, fim, lpf, saf, stb, stf, and STM4595) which is common between Salmonella serovars and responsible for colonization of  host cells. (Huehn et al., 2009).

The sopB gene associated with prophages was found in 92.3% of the examined isolates. Different studies have also reported the detection of that gene in almost all the Salmonella isolates from food and human origin (Borges et al., 2013; Ahmed et al., 2016).

There are other genes on prophage may have a role in virulence as the prophages Gifsy1, 2, and 3, Fels-1 and 2, and SopEF (Ehrbar and Hardt, 2005). The SPI-1 secreted effectors SopE and SopE2 act as guanine nucleotide-exchange-factors (GEFs) for the small GTPases Cdc42 and Rac (Thomson et al. 2004) in present study was detected by 19% which is nearly low as in (Osman et al., 2014b).

SodC found in pathogenic Gram-negative and Gram-positive bacteria (Sanjay et al., 2010). It is responsible for protecting the pathogens against superoxide radicals generated by inflammatory and phagocytic cells during infections has been emphasized, non-detection of sodC may be due to their low expression and/or the instability of the enzyme due to proteolysis (Sanjay et al., 2010).

The virulence plasmid gene spvC was detected in the lowest percent among other virulence genes (Huehn et al., 2010).

gipA was absent in all strains as found in (Osman et al., 2014). gipA, is stimulated when the bacteria colonize the small intestine, after infection takes place several genes are elicited due to bacterial growth in Peyer's patch in small intestine (Stanley  et al., 2000).

This study shows that  virulence genes are widely distributed among Salmonella Kentucky which may pose as potential  risk for poultry and  human infections. Virulence genes are located on transmissible genetic elements as transposons, plasmids or bacteriophages or pathogenicity islands (Hacker et al., 1997).

In conclusion, the presence of these entire virulence gene in Salmonella Kentucky explain the increase of rate of isolation of this serotype from human and animals allover the world.

ACKNOWLEDGEMENTS

We are very grateful to Omneya sarea, Abdelhafez Samir for their  technical help.

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